Mitochondrial function deteriorates with aging. We have shown that aging is associated with decreased mitochondrial protein, mitochondrial oxidative phosphorylation, complex III (CIII) and IV activity, and is associated with decrease in major cardiolipin species [CL(C18:2)4] and increase in docosahexaenoic acid containing CL [CL(C18:2)3, (C22:6)1]. The activity of complex IV is restored in the presence of phospholipids. These age-associated changes are observed in interfibrillar (IFM) mitochondria, but not in subsarcolemmal mitochondria (SSM). Because the activity of mitochondrial electron transport chain complexes is profoundly affected by the lipid environment of the mitochondrial inner membrane, specifically CL, we proposed that there is increased CL turnover/remodeling. To experimentally test our hypothesis we will use a novel heavy water (2H2O) metabolic labeling technique we have developed for a global lipidome and proteome turnover studies in free living animals. In Aim 1 we determine the effect of aging on phospholipid kinetics in SSM and IFM from 6 and 24 months old rats with particular focus on the turnover of the distinctive CL species [CL(C18:2)3, (C22:6)1] specific for mitochondria of aged rats. The glycerol moiety of CL will be used to assess CL biosynthesis and the turnover of minor nonessential fatty acids in the 6 month and docosahexaenoic acid in the 24 month old animals. Linoleic acid, an essential fatty acid, will not be labeled, which has been a problem in the two published studies. In contrast, we propose to use the glycerol moiety, which will be labeled and is the backbone while the fatty acids are part of the remodeling. In Aim 2 we determine the turnover and remodeling of CL associated with free CIII and CIII incorporated into supercomplexes in IFM in comparison to SSM, which are unaffected by aging. Is CL kinetics in cardiac IFM from aged rats restored to that observed in SSM and to IFM for 6 month old animals following restoration of complex III activity with acetyl-carnitine? Does the turnover of CIII protein, in specific cytochrome b, increase with restoration of CIII activity and of mitochondrial oxidative phosphorylation in IFM? The goal of this R21 is to collect preliminary data relevant to the mechanism of age-associated decreased mitochondrial function by determining the turnover of mitochondrial phospholipids and CL in specific using a novel heavy water (2H2O) metabolic labeling technique we have developed for a global proteome turnover studies in free living animals. Application of this novel approach to assess the effect of aging on mitochondrial function will provide valuable insight into the molecular mechanism of cardiac aging. The understanding gained from our proposed work will foster the development of strategies to counter the negative impact of aging on cardiac metabolism and to delay or slow the rate of cardiovascular mortality and morbidity.